Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

Xiaoqing Zhang,Peter Arvan

doi:10.1074/jbc.m003559200

Abstract

Recently, it has been suggested that only approximately 2% of human thyroid peroxidase (hTPO(933)) reaches the surface of stably transfected (Chinese hamster ovary) cells, most being degraded intracellularly, and this might be representative of thyroid peroxidase (TPO) behavior in thyrocytes (Fayadat, L., Siffroi-Fernandez, S., Lanet, J., and Franc, J.-L. (2000) J. Biol. Chem. 275, 15948-15954). In agreement, in stably transfected Madin-Darby canine kidney clones, nonpermeabilized cells exhibit wild-type hTPO(933) immunofluorescence (apically) on <10% of that found in permeabilized cells, where an endoplasmic reticulum pattern is observed. Further, a C-terminally truncated, membrane-anchorless hTPO(848) is also retained in the endoplasmic reticulum of stably transfected Madin-Darby canine kidney cells. However, by contrast, in Chinese hamster ovary cells after transient transfection, hTPO(933) immunofluorescence is detected equally well in nonpermeabilized and permeabilized cells, indicating that a large portion of hTPO(933) is present at the cell surface; furthermore, hTPO(848) is efficiently secreted. Further, using an antiserum not cross-reacting with rat TPO, we find by immunofluorescence that in stable clones of PC Cl3 (rat) thyrocytes, considerably more ( approximately 50%) of the cells exhibit hTPO(933) at the cell surface. However, cell surface biotinylation and endoglycosidase H digestion assays appear to under-represent the extent of hTPO(933) transport, presumably because protein folding limits both Golgi carbohydrate modification and accessibility of lysines in the extracellular domain. We conclude that cell type-specific factors may facilitate stable expression of TPO at the cell surface of thyrocytes.

Highlights

In the past decade, cell type-dependent differences in membrane and secretory protein trafficking have been increasingly recognized
It has been suggested that only ϳ2% of human thyroid peroxidase reaches the surface of stably transfected (Chinese hamster ovary) cells, most being degraded intracellularly, and this might be representative of thyroid peroxidase (TPO) behavior in thyrocytes (Fayadat, L., Siffroi-Fernandez, S., Lanet, J., and Franc, J.-L. (2000) J
The original reports of recombinant hTPO expression indicated the presence of a subpopulation of antibiotic-resistant CHO cells in which stably expressed, full-length wild-type hTPO933 was clearly detected on the plasma membrane [31, 32] and a truncated hTPO848 was efficiently secreted into the medium [33,34,35]; both of these recombinant hTPO constructs were proven to function as enzymatically active peroxidases

Summary

Introduction

Cell type-dependent differences in membrane and secretory protein trafficking have been increasingly recognized. The original reports of recombinant hTPO expression indicated the presence of a subpopulation of antibiotic-resistant CHO cells in which stably expressed, full-length wild-type hTPO933 was clearly detected on the plasma membrane [31, 32] and a truncated hTPO848 (mutated to place a stop codon that eliminates the transmembrane span and cytosolic tail, as in Fig. 1) was efficiently secreted into the medium [33,34,35]; both of these recombinant hTPO constructs were proven to function as enzymatically active peroxidases.

Results

Conclusion